Acute cell-mediated rejection in humans, a major cause of early graft failure and a risk for increased late graft failure, is mediated by circulatin host effector memory (EM) T cells that infiltrate into the graft where they proliferate and differentiate into the effector T cells that are responsible for graft injury and loss. The infiltrtion takes place through post-capillary venules made up of a monolayer of graft-derived endothelial cells (EC) lining a structure supported by graft-derived pericytes (PC) and T cell proliferation/differentiation likely takes place within the infiltrates that initially accumulate aound the microvessels in proximity to the PC. Our research uniquely focuses on how human EC and PC contribute to these processes. In specific aim 1 of this competitive renewal, we will extend our highly productive investigation of how direct recognition of allogeneic MHC molecules displayed by human EC contributes to recruitment of EM CD4+ T cells to EM CD8+ T cells, EMRA CD8+ T cells and CD4+ regulatory T cells (T regs), and how transmigration through EC, whether triggered by chemokine or TCR engagement, influences subsequent T cell activation and differentiation. We will use both cell culture models under realistic conditions of flow and in vivo models involving the formation of human EC- lined microvessels that have been implanted and are perfused in immunodeficient mice. We will supplement conventional antibody blocking approaches with novel gene editing of human EC and protein transfection of human T cells. In aim 2, we will use human PC, isolated from placenta and skin, to examine their effects on recruitment, activation and differentiation of the same T cell subsets, alone and in combination with EC, again using novel models that we have developed. We believe that our identification of a significantly understudied issue of great importance, our track record of accomplishments, and our innovative approaches set us apart from all other laboratories studying transplant rejection. The results of our studies may open the way to new therapies that target the vasculature of the graft rather than the immune system of the host, reducing toxicity and morbidity.